Oxygen Activation and Arene Hydroxylation byFunctional Mimics of r-Keto Acid-DependentIron(II) Dioxygenases

Eric L. Hegg, Raymond Y. N. Ho, and Lawrence Que, Jr.*

Department of Chemistry and Center forMetals in Biocatalysis

UniVersity of Minnesota, Minneapolis, Minnesota 55455

ReceiVed NoVember 9, 1998

R-Keto acid-dependent enzymes, which represent the largestand most diverse class of non-heme iron enzymes, utilize anR-keto acid cofactor to effect dioxygen activation in numerousbiochemical pathways (Scheme 1).1,2 Crystallographic studies ofone member of this class, deacetoxycephalosporin C synthase(DAOCS),3 reveal a 2-His-1-carboxylate facial triad at the ironcenter, an emerging motif among mononuclear nonheme ironenzymes.4 In the enzyme-cofactor complex, theR-ketoglutarate(R-KG) cofactor is chelated to the iron through the carboxylateand theR-keto oxygen. These crystal structures confirm the modelpreviously proposed based on various spectroscopic and bio-chemical studies.2,5 While the few model iron(II)-R-keto acidcomplexes available6,7 have provided important mechanisticinsight, they have not demonstrated the dioxygenase reactivityexhibited by the enzymes. Here we report the structure andproperties of [Fe(TpPh2)(BF)] (1),8 which reacts with O2 to effectintramolecular arene hydroxylation, incorporating both atoms ofO2 into the products and thus demonstrating for the first time thedioxygenase activity exhibited byR-keto acid-dependent enzymes.In addition, the reactivity of1 is contrasted with that of [Fe-(TpPh2)(OBz)], emphasizing the unique function of theR-keto acidcofactor in oxygen activation.

Complex 1 was synthesized by the anaerobic, sequentialaddition of equimolar amounts of FeCl2, K(TpPh2),9 and theR-ketoacid sodium benzoylformate (BF) in an acetonitrile slurry(Supporting Information). The crystal structure of110 (Figure 1)reveals a 5-coordinate iron(II) center with a monoanionic, face-capping TpPh2 and a chelated BF, similar to that of the relatedcomplex [Fe(Tpt-Bu,i-Pr)(BF)];11 two pyrazole nitrogens and acarboxylate oxygen define the equatorial plane of the distortedtrigonal bipyramid (τ ) 0.65).12 The optical spectrum of1 (Figure2A) exhibits an absorption band at 531 nm (340 M-1cm-1) withshoulders at 476 (210 M-1 cm-1) and 584 nm (300 M-1 cm-1).This feature, which is diagnostic of anR-keto acid chelated toan iron(II) center through theR-keto and one carboxylate oxygen,is blue-shifted (Figure 2B) when BF is replaced with an aliphaticR-keto acid, pyruvate (Phf Me), consistent with its assignmentto MLCT transitions.5,6,11

(1) Abbott, M. T.; Udenfriend, S. InMolecular Mechanisms of OxygenActiVation; Hayaishi, O., Ed.; Academic Press: New York, 1974; pp 167-214.

(2) Que, L., Jr.; Ho, R. Y. N.Chem. ReV. 1996, 96, 2607-2624.(3) Valegard, K.; van Scheltinga, A. C. T.; Lloyd, M. D.; Hara, T.;

Ramaswamy, S.; Perrakis, A.; Thompson, A.; Lee, H.-J.; Baldwin, J. E.;Schofield, C. J.; Hajdu, J.; Andersson, I.Nature1998, 394, 805-809.

(4) Hegg, E. L.; Que, L., Jr.Eur. J. Biochem.1997, 250, 625-629.(5) Pavel, E. G.; Zhou, J.; Busby, R. W.; Gunsior, M.; Townsend, C. A.;

Solomon, E. I.J. Am. Chem. Soc.1998, 120, 743-753.(6) Chiou, Y.-M.; Que, L., Jr.J. Am. Chem. Soc.1995, 117, 3999-4013.(7) Ha, E. H.; Ho, R. Y. N.; Kisiel, J. F.; Valentine, J. S.Inorg. Chem.

1995, 34, 2265-2266.(8) Abbreviations: BF, benzoylformate; DAOCS, deacetoxycephalosporin

C synthase;R-KG, R-ketoglutarate; MLCT, metal-to-ligand charge transfer;Tpt-Bu,i-Pr, hydrotris(3-tert-butyl-5-isopropylpyrazol-1-yl)borate; TpMe2, hy-drotris(3,5-dimethylpyrazol-1-yl)borate; TpPh2, hydrotris(3,5-diphenylpyrazol-1-yl)borate.

(9) Kitajima, N.; Fujisawa, K.; Fujimoto, C.; Moro-oka, Y.; Hashimoto,S.; Kitagawa, T.; Toriumi, K.; Tatsumi, K.; Nakamura, A.J. Am. Chem. Soc.1992, 114, 1277-1291.

(10) X-ray quality violet needles were obtained over a period of severalweeks from an acetone solution at-20 °C. 1 crystallizes in the monoclinicspace groupP21, with a ) 10.0654(3) Å,b ) 21.2126(5) Å,c ) 12.0796(3)Å, â ) 97.307(1)°, V ) 2558.21(12) Å3, andZ ) 2. The structure was solvedand refined with SHELXTL 5.0 (Siemens Industrial Automation: Madison,WI). Two molecules of acetone cocrystallized with each molecule of1. 7996unique reflections were collected to 25.07° 2θ at 173 K with Mo KR (λ )0.71073 Å radiation) on a Siemens SMART Platform CCD. The structurewas solved by direct methods and refined toR ) 0.0713 andRw ) 0.1083 (I> 2σ(I) ) 4890).

(11) Hikichi, S.; Ogihara, T.; Fujisawa, K.; Kitajima, N.; Akita, M.; Moro-oka, Y. Inorg. Chem.1997, 36, 4539-4547.

(12) According to this formalism, a perfect square pyramid would have aτ-value of 0.00, while a perfect trigonal bipyramid would have aτ-value of1.00 (Addison, A. W.; Rao, T. N.; Reedijk, J.; van Rijn, J.; Verschoor, G. C.J. Chem. Soc., Dalton Trans.1984, 1349-1356).

(13) In a typical experiment, 1 mg of1 was dissolved in 1 mL of anaerobic,distilled benzene and then diluted with an additional 2 mL of O2-saturatedbenzene.

Scheme 1

Figure 1. X-ray structure of [Fe(TpPh2)(BF)] (1) showing 30% probabilitythermal ellipsoids. Hydrogen atoms and two acetone solvent moleculesare omitted for clarity. Selected bond lengths (Å) and distances (deg)are: Fe-O1, 1.968(4); Fe-O2, 2.206(5); Fe-N2, 2.188(5); Fe-N4,2.068(5); Fe-N6, 2.068(5); O1-Fe-O2, 77.3(2); N2-Fe-N4, 86.4(2);N2-Fe-N6, 89.0(2); N4-Fe-N6, 91.1(2); O1-Fe-N2, 110.1(2); O2-Fe-N2, 171.7(2); O1-Fe-N4, 132.6(2); O2-Fe-N4, 85.7(2); O1-Fe-N6, 131.5(2); O2-Fe-N6, 88.5(2).

Figure 2. Optical spectra of (A) a 0.25 mM solution of1 (R ) Ph), (B)a 0.25 mM solution of the corresponding pyruvate complex (R) Me),and (C) the product3 formed from the reaction of 0.25 mM1 with excessO2. Inset: resonance Raman spectra of3 obtained from frozen CH2Cl2solutions with 632.8 nm excitation.

1972 J. Am. Chem. Soc.1999,121,1972-1973

10.1021/ja983867u CCC: $18.00 © 1999 American Chemical SocietyPublished on Web 02/13/1999

Complex1 reacts with O2 over the course of 30 min13 affordingthe quantitative decarboxylation of BF to benzoic acid.14 Con-comitant with this transformation is the generation of a greenspecies (3) (λmax ) 650 nm, Figure 2C) whose resonance Ramanspectrum (Figure 2 inset) exhibits features characteristic of aniron(III) coordinated to an ortho-substituted phenolate.15 Theidentical product is also obtained when [Fe(TpPh2)(OBz)] isexposed to O2, but the reaction takes approximately 14 h. Theanalogous green species generated from the reaction of O2 with[Fe(TpPh,Me)(OOCR)] has been identified by Fujisawa et al. to bean iron(III) complex in which one of the 3-phenyl groups of theTp ligand is hydroxylated.16 Combined, these results demonstratethat 1 reacts with O2 to form 2, which autoxidizes to green3(Scheme 2).

The fate of dioxygen was established by18O2-labeling experi-ments (95%, Cambridge Isotope). Analysis of the OBz productvia GC-mass spectrometry demonstrated that approximately 80%of the newly formed carboxylate contained one atom of oxygenfrom O2. The substoichiometric incorporation is consistent withpreviously published results and is attributed to a competingautoxidation reaction.6 The other18O atom is incorporated intothe phenolate product as evidenced by the diagnostic shifts inkey peak frequencies in the resonance Raman spectrum.17,18

Incorporation of the label into the phenolate is nearly quantitative(90%) as deduced from the intensity of the residual 621 cm-1

peak (νFe-16OAr) relative to that of the 605 cm-1 peak (νFe-18OAr)(Figure 2 inset). Thus,1 reproduces the dioxygenase nature of

R-keto acid-dependent enzymes (Scheme 1) and, in particular,mimics p-hydroxyphenylpyruvate dioxygenase in its ability tohydroxylate a phenyl ring.19

Steric effects play a key role in modulating the reactivity ofR-keto acid complexes. Whereas1 reacts with O2 within 30 min,the sterically encumbered [Fe(Tpt-Bu,i-Pr)(BF)] is completelyunreactive toward O2.11 Consistent with this trend, [Fe(TpMe2)-(BF)] reacts with O2 within minutes.7 Furthermore, when thephenyl group of the BF moiety on1 is replaced with Me ori-Pr,20

the resulting complexes effect arene hydroxylation significantlyfaster. The order of reactivity for the different R groups is Ph<i-Pr < Me, with R ) Me reacting almost an order of magnitudefaster than BF. This is the opposite of what is expected based onprevious results where electron-withdrawing groups on ring-substituted BFs enhanced reactivity, which was consistent witha partially rate-limiting nucleophilic attack of an Fe-boundsuperoxide on theR-carbon.6 Taken together, these results indicatethat steric constraints are an important determinant of the reactivityand suggest that the transformation of the O2 adducts into productsentails molecular motions that require ample space around theiron center.

The fact that both1 and [Fe(TpPh2)(OBz)] react with O2 toproduce3 suggests that these complexes generate related, if notidentical, oxidizing agents during the course of the reaction. Anattractive intermediate is a [(TpPh2)FeIVdO] species. The 30-folddifference in reactivity between these two complexes (0.5 h vs14 h) thus can be rationalized by the differing mechanisms eachutilizes to form the putative oxene species. [Fe(TpPh2)(OBz)] verylikely generates the iron-oxo species via O-O bond lysis of a(µ-1,2-peroxo)diiron(III) intermediate, which has previously beenobserved and crystallized for a Tpi-Pr2 derivative.21,22 Complex1, on the other hand, is proposed to form the iron-oxo speciesvia lysis of a mononuclear iron-peroxo species, derived fromnucleophilic attack of the FeIII -bound superoxide onto theR-carbon of the keto acid (Scheme 2).6 Significantly, the activationenergy for O-O bond cleavage may be lowered considerably bycoupling this step to the loss of CO2. Thus, 1 effects arenehydroxylation significantly faster than [Fe(TpPh2)(OBz)], high-lighting the importance of theR-keto acid in activating O2.

In conclusion, [Fe(TpPh2)(BF)] is both a structural and func-tional mimic of R-keto acid-dependent enzymes. The mono-anionic, tridentate, facially capping TpPh2ligand approximates theendogenous 2-His-1-carboxylate facial triad found in DAOCS,BF chelates to the iron center, and an available coordination siteis maintained for binding O2. Upon exposure of1 to O2, BFundergoes oxidative decarboxylation, and the TpPh2 ligand ishydroxylated, with 1 atom of oxygen being incorporated into eachproduct, mimicking for the first time the dioxygenase nature ofthe enzymatic reaction. The use of theR-keto acid cofactor thusallows these enzymes to activate dioxygen and generate a versatileoxidant via a mechanism which is distinct from those of enzymesthat require a porphyrin ring23 or a second metal ion.24

Acknowledgment. The authors thank Professor Kiyoshi Fujisawa(Tokyo Institute of Technology) for sharing unpublished results and Dr.Victor G. Young, Jr. (University of Minnesota) for solving the crystalstructure of1. This work has been supported by the National Institutesof Health (GM33162), and NIH postdoctoral fellowship support for E.L.H.(GM18639) and R.Y.N.H. (GM17849) is gratefully acknowledged.

Supporting Information Available: Further experimental detailsconcerning the synthesis and characterization of1, [Fe(TpPh2)(pyruvate)],[Fe(TpPh2)(O2CC(O)CH(CH3)2)], and [Fe(TpPh2)(OBz)], full resonanceRaman spectra of3, and tables of crystal data, data collection, structuresolution and refinement, atomic coordinates, and bond lengths and anglesfor 1 are available (PDF). This material is available free of charge viathe Internet at http://pubs.acs.org.

JA983867U

(14) Benzoate was formed stoichiometrically as determined via GC.Quantification entailed decomposing the inorganic complex with 0.1 M H2-SO4, extracting the organic products with Et2O and CHCl3, and esterifyingwith diazomethane according to standard procedures. Methyl 3-chlorobenzoatewas added after degradation of the complex as an internal standard.

(15) Carrano, C. J.; Carrano, M. W.; Sharma, K.; Backes, G.; Sanders-Loehr, J.Inorg. Chem.1990, 29, 1865-1870.

(16) Fujisawa, K.; Suezaki, M.; Moro-oka, Y., submitted for publication.(17) Raman frequencies (cm-1) of dioxygen-sensitive peaks of sample

prepared with16O2 or (18O2): 621 (605); 861 (844); 961 (942); 1254 (1245);and 1298 (1294).

(18) Pyrz, J. W.; Roe, A. L.; Stern, L. J.; Que, L., Jr.J. Am. Chem. Soc.1985, 107, 614-620.

(19) Lindblad, B.; Lindstedt, G.; Lindstedt, S.J. Am. Chem. Soc.1970,92, 7446-7449.

(20) These complexes were synthesized in a manner analogous to that of1 except that Fe(ClO4)2 was utilized in place of FeCl2 and the reaction wasperformed in MeOH. The insoluble products were collected by filtration.

(21) Kim, K.; Lippard, S. J.J. Am. Chem. Soc.1996, 118, 4914-4915.(22) Kitajima, N.; Tamura, N.; Amagai, H.; Fukui, H.; Moro-oka, Y.;

Mizutani, Y.; Kitagawa, T.; Mathur, R.; Heerwegh, K.; Reed, C. A.; Randall,C. R.; Que, L., Jr.; Tatsumi, K.J. Am. Chem. Soc.1994, 116, 9071-9085.

(23) Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H.Chem. ReV.1996, 96, 2841-2887.

(24) Wallar, B. J.; Lipscomb, J. D.Chem. ReV. 1996, 96, 2625-2657.

Scheme 2

Communications to the Editor J. Am. Chem. Soc., Vol. 121, No. 9, 19991973